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The Trypanosoma brucei genome is structurally complex. Eleven megabase-sized chromosomes each comprise a transcribed core flanked by silent subtelomeres, housing thousands of Variant Surface Glycoprotein (VSG) genes. Additionally, hundreds of sub-megabase chromosomes contain 177 bp repeats of unknown function, and VSG transcription sites localise to many telomeres. DNA replication dynamics have only been described in the megabase chromosome cores, and in the single active VSG transcription site. Using a Nanopore genome assembly, we show that megabase chromosome subtelomeres display a paucity of replication initiation events relative to the core, correlating with increased instability. In addition, replication of the active VSG transcription site is shown to originate from the telomere, likely causing targeted VSG recombination. Lastly, we provide evidence that the 177 bp repeats act as conserved DNA replication origins, explaining submegabase chromosome stability. Compartmentalized DNA replication therefore explains how T. brucei balances stable genome transmission with localised instability driving immune evasion., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The rate and pattern of mutagenesis in cancer genomes is significantly influenced by DNA accessibility and active biological processes. Here we show that efficient sites of replication initiation drive and modulate specific mutational processes in cancer. Sites of replication initiation impede nucleotide excision repair in melanoma and are off-targets for activation-induced deaminase (AICDA) activity in lymphomas. Using ductal pancreatic adenocarcinoma as a cancer model, we demonstrate that the initiation of DNA synthesis is error-prone at G-quadruplex-forming sequences in tumours displaying markers of replication stress, resulting in a previously recognised but uncharacterised mutational signature. Finally, we demonstrate that replication origins serve as hotspots for genomic rearrangements, including structural and copy number variations. These findings reveal replication origins as functional determinants of tumour biology and demonstrate that replication initiation both passively and actively drives focal mutagenesis in cancer genomes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

DNA replication represents a series of precisely regulated events performed by a complex protein machinery that guarantees accurate duplication of the genetic information. Since DNA replication is permanently faced by a variety of exogenous and endogenous stressors, DNA damage response, repair and replication must be closely coordinated to maintain genomic integrity. HROB has been identified recently as a binding partner and activator of the Mcm8/9 helicase involved in DNA interstrand crosslink (ICL) repair. We identified HROB independently as a nuclear protein whose expression is co-regulated with various DNA replication factors. Accordingly, the HROB protein level showed a maximum in S phase and a downregulation in quiescence. Structural prediction and homology searches revealed that HROB is a largely intrinsically disordered protein bearing a helix-rich region and a canonical oligonucleotide/oligosaccharide-binding-fold motif that originated early in eukaryotic evolution. Employing a flow cytometry Förster resonance energy transfer (FRET) assay, we detected associations between HROB and proteins of the DNA replication machinery. Moreover, ectopic expression of HROB protein led to an almost complete shutdown of DNA replication. The available data imply a function for HROB during DNA replication across barriers such as ICLs.

TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungus Ustilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed that UmTrf2 binds to Blm in vitro and inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activity in vivo, over-expression of Trf2 induces telomere shortening, just like deletion of blm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion of blm or mre11 (but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably, U. maydis harbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion of tay1 partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Syed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

DNA replication is a complex and crucial biological process in eukaryotes. To facilitate the study of eukaryotic replication events, we present a database of eukaryotic DNA replication origins (DeOri), which collects genome-wide data on eukaryotic DNA replication origins currently available. With the rapid development of high-throughput experimental technology in recent years, the number of datasets in the new release of DeOri 10.0 increased from 10 to 151 and the number of sequences increased from 16,145 to 9,742,396. Besides nucleotide sequences and browser extensible data (BED) files, corresponding annotation files, such as coding sequences (CDSs), mRNAs, and other biological elements within replication origins, are also provided. The experimental techniques used for each dataset, as well as related statistical data, are also presented on web page. Differences in experimental methods, cell lines, and sequencing technologies have resulted in distinct replication origins, making it challenging to differentiate between cell-specific and non-specific replication origins. Based on multiple replication origin datasets at the species level, we scored and screened replication origins in Homo sapiens, Gallus gallus, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans. The screened regions with high scores were considered as species-conservative origins, which are integrated and presented as reference replication origins (rORIs). Additionally, we analyzed the distribution of relevant genomic elements associated with replication origins at the genome level, such as CpG island (CGI), transcription start site (TSS), and G-quadruplex (G4). These analysis results can be browsed and downloaded as needed at http://tubic.tju.edu.cn/deori/., (© The Author(s) 2024. Published by Oxford University Press and Science Press on behalf of the Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China.)

Completion of DNA replication before chromosome segregation is essential for the stable maintenance of the genome. Under replication stress, DNA synthesis may persist beyond S phase, especially in genomic regions that are difficult to proceed with the replication processes. Incomplete replication in mitosis emerges as non-disjoined segment in mitotic chromosomes leading to anaphase bridges. The resulting chromosome rearrangements are not well characterized, however. Here, we report that incomplete replication due to SMC5/6 deficiency impairs sister chromatid disjunction at difficult-to-replicate regions, including common fragile sites. These non-disjoined regions manifest as cytologically defined symmetric gaps, causing anaphase bridges. These bridges break at the gaps, leading to telomere loss, micronucleation, and fragmentation. Subsequently, fusions between telomere-deficient chromosomes generate complex chromosomal rearrangements, including dicentric chromosomes, suggesting the occurrence of breakage-fusion-bridge cycle. Additionally, chromosomes in micronuclei were pulverized, indicative of chromothripsis. Our findings suggest that incomplete replication facilitates complex chromosomal rearrangements, which may contribute to genomic instability in human cancers., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Locations of DNA replication initiation in prokaryotes, called "origins of replication", are well-characterized. However, a mechanistic understanding of the sequence dependence of the local unzipping of double-stranded DNA, the first step towards replication initiation, is lacking. Here, utilizing a Markov chain model that was created to address the directional nature of DNA unzipping and replication, we model the sequence dependence of local melting of double-stranded linear DNA segments. We show that generalized palindromic sequences with high nucleotide skews have a low kinetic barrier for local melting near melting temperatures. This allows for such sequences to function as potential replication origins. We support our claim with evidence for high-skew palindromic sequences within the replication origins of mitochondrial DNA, bacteria, archaea and plasmids., Competing Interests: Declarations. Conflicts of interest: The authors declare no conflict of interest., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background/objectives: Nucleolin is a major component of the nucleolus and is involved in various aspects of ribosome biogenesis. However, it is also implicated in non-nucleolar functions such as cell cycle regulation and proliferation, linking it to various pathologic processes. The aim of this study was to use differential gene expression analysis and Weighted Gene Co-expression Network analysis (WGCNA) to identify nucleolin-related regulatory pathways and possible key genes as novel therapeutic targets for cancer, viral infections and other diseases., Methods: We used two different siRNAs to downregulate the expression of nucleolin in a human hepatoblastoma (HepG2) cell line. We carried out RNA-sequencing (RNA-Seq), performed enrichment analysis of the pathways of the differentially expressed genes (DEGs) and identified protein-protein interaction (PPI) networks., Results: Both siRNAs showed high knockdown efficiency in HepG2 cells, resulting in the disruption of the nucleolar architecture and the downregulation of rRNA gene expression, both downstream hallmarks of a loss of nucleolin function. RNA-Seq identified 44 robust DEGs in both siRNA cell models. The enrichment analysis of the pathways of the downregulated genes confirmed the essential role of nucleolin in DNA replication and cell cycle processes. In addition, we identified seven hub genes linked to NCL : MCM6 , MCM3 , FEN1 , MYBL2 , MSH6 , CDC6 and RBM14 ; all are known to be implicated in DNA replication, cell cycle progression and oncogenesis., Conclusions: Our findings demonstrate the functional consequences of nucleolin depletion in HepG2 and confirm the importance of nucleolin in DNA replication and cell cycle processes. These data will further enhance our understanding of the molecular and pathologic mechanisms of nucleolin and provide new therapeutic perspectives in disease.

The evolution of operons has puzzled evolutionary biologists since their discovery, and many theories exist to explain their emergence, spreading, and evolutionary conservation. In this work, we suggest that DNA replication introduces a selective force for the clustering of functionally related genes on chromosomes, which we interpret as a preliminary and necessary step in operon formation. Our reasoning starts from the observation that DNA replication produces copy number variations of genomic regions, and we propose that such changes perturb metabolism. The formalization of this effect by exploiting concepts from metabolic control analysis suggests that the minimization of such perturbations during evolution could be achieved through the formation of gene clusters and operons. We support our theoretical derivations with simulations based on a realistic metabolic network, and we confirm that present-day genomes have a degree of compaction of functionally related genes, which is significantly correlated to the proposed perturbations introduced by replication. The formation of clusters of functionally related genes in microbial genomes has puzzled microbiologists since their first discovery. Here, we suggest that replication, and the copy number variations due to the replisome passage, might play a role in the process through a perturbation in metabolite homeostasis. We provide theoretical support to this hypothesis, and we found that both simulations and genomic analysis support our hypothesis., Importance: The formation of clusters of functionally related genes in microbial genomes has puzzled microbiologists since their discovery. Here, we suggest that replication, and the copy number variations due to the replisome passage, might play a role in the process through a perturbation in metabolite homeostasis. We provide theoretical support to this hypothesis, and we found that both simulations and genomic analysis support our hypothesis., Competing Interests: The authors declare no conflict of interest.

Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). The coordinated segregation of the replication terminus of both chromosomes (TerI and TerII) determines when and where cell division occurs. ChrI encodes a homologue of Escherichia coli MatP, a protein that binds to a DNA motif (matS) that is overrepresented in replication termini. Here, we use a combination of deep sequencing and fluorescence microscopy techniques to show that V. cholerae MatP structures TerI and TerII into macrodomains, targets them to mid-cell during replication, and delays their segregation, thus supporting that ChrII behaves as a bona fide chromosome. We further show that the extent of the segregation delay mediated by MatP depends on the number and local density of matS sites, and is independent of its assembly into tetramers and any interaction with the divisome, in contrast to what has been previously observed in E. coli., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA sequencing (RNA-seq) analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range downregulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA-DNA hybrid is a part of the R-loop which is an important non-standard nucleic acid structure. RNA-DNA hybrid/R-loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA-DNA hybrids and map their genomic locations in fission yeast cells. The RNA-DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild-type. The majority of RNA-DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S-phase and about 20% of the RNA-DNA hybrids overlap with Rad52 loci. During S-phase, more robust association of Rad52 with RNA-DNA hybrids was observed in the protein coding region than in M-phase. These results suggest that persistent RNA-DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S-phase, potentially through collision with DNA replication forks., (© 2024 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Homologous recombination (HR) factors are crucial for DSB repair and processing stalled replication forks. RAD51 paralogs, including RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3, have emerged as essential tumour suppressors, forming two subcomplexes, BCDX2 and CX3. Mutations in these genes are associated with cancer susceptibility and Fanconi anaemia, yet their biochemical activities remain unclear. This study reveals a linear arrangement of BCDX2 subunits compared to the RAD51 ring. BCDX2 shows a strong affinity towards single-stranded DNA (ssDNA) via unique binding mechanism compared to RAD51, and a contribution of DX2 subunits in binding branched DNA substrates. We demonstrate that BCDX2 facilitates RAD51 loading on ssDNA by suppressing the cooperative requirement of RAD51 binding to DNA and stabilizing the filament. Notably, BCDX2 also promotes RAD51 loading on short ssDNA and reversed replication fork substrates. Moreover, while mutants defective in ssDNA binding retain the ability to bind branched DNA substrates, they still facilitate RAD51 loading onto reversed replication forks. Our study provides mechanistic insights into how the BCDX2 complex stimulates the formation of BRCA2-independent RAD51 filaments on short stretches of ssDNA present at ssDNA gaps or stalled replication forks, highlighting its role in genome maintenance and DNA repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

MacroH2A has been linked to transcriptional silencing, cell identity, and is a hallmark of the inactive X chromosome (Xi). However, it remains unclear whether macroH2A plays a role in DNA replication. Using knockdown/knockout cells for each macroH2A isoform, we show that macroH2A-containing nucleosomes slow down replication progression rate in the Xi reflecting the higher nucleosome stability. Moreover, macroH2A1, but not macroH2A2, regulates the number of nano replication foci in the Xi, and macroH2A1 downregulation increases DNA loop sizes corresponding to replicons. This relates to macroH2A1 regulating replicative helicase loading during G1 by interacting with it. We mapped this interaction to a phenylalanine in macroH2A1 that is not conserved in macroH2A2 and the C-terminus of Mcm3 helicase subunit. We propose that macroH2A1 enhances the licensing of pre-replication complexes via DNA helicase interaction and loading onto the Xi., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The cell cycle-regulated DNA methyltransferase CcrM is conserved in most Alphaproteobacteria, but its role in bacteria with complex or multicentric genomes remains unexplored. Here, we compare the methylome, the transcriptome and the phenotypes of wild-type and CcrM-depleted Agrobacterium tumefaciens cells with a dicentric chromosome with two essential replication origins. We find that DNA methylation has a pleiotropic impact on motility, biofilm formation and viability. Remarkably, CcrM promotes the expression of the repABCCh2 operon, encoding proteins required for replication initiation/partitioning at ori2, and represses gcrA, encoding a conserved global cell cycle regulator. Imaging ori1 and ori2 in live cells, we show that replication from ori2 is often delayed in cells with a hypo-methylated genome, while ori2 over-initiates in cells with a hyper-methylated genome. Further analyses show that GcrA promotes the expression of the RepCCh2 initiator, most likely through the repression of a RepECh2 anti-sense RNA. Altogether, we propose that replication at ori1 leads to a transient hemi-methylation and activation of the gcrA promoter, allowing repCCh2 activation by GcrA and contributing to initiation at ori2. This study then uncovers a novel and original connection between CcrM-dependent DNA methylation, a conserved epigenetic regulator and genome maintenance in an Alphaproteobacterial pathogen., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanism for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrate that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we show that targeting dCas9 to telomeres using sgTelo impedes DNA replication at telomeres and induces a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigate the genome-wide features of telomeres in the dCas9/sgTelo cells and observe a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-. Consistently, we also observe an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncover many interesting molecular and structural features of the ITCB and demonstrate that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Replication, heredity, and evolution are characteristic of Life. We and others have postulated that the reconstruction of a synthetic living system in the laboratory will be contingent on the development of a genetic self-replicator capable of undergoing Darwinian evolution. Although DNA-based life dominates, the in vitro reconstitution of an evolving DNA self-replicator has remained challenging. We hereby emulate in liposome compartments the principles according to which life propagates information and evolves. Using two different experimental configurations supporting intermittent or semi-continuous evolution (i.e., with or without DNA extraction, PCR, and re-encapsulation), we demonstrate sustainable replication of a linear DNA template - encoding the DNA polymerase and terminal protein from the Phi29 bacteriophage - expressed in the 'protein synthesis using recombinant elements' (PURE) system. The self-replicator can survive across multiple rounds of replication-coupled transcription-translation reactions in liposomes and, within only ten evolution rounds, accumulates mutations conferring a selection advantage. Combined data from next-generation sequencing with reverse engineering of some of the enriched mutations reveal nontrivial and context-dependent effects of the introduced mutations. The present results are foundational to build up genetic complexity in an evolving synthetic cell, as well as to study evolutionary processes in a minimal cell-free system., (© 2024. The Author(s).)

The cell cycle of budding yeast is governed by an intricate protein regulatory network whose dysregulation can lead to lethal mistakes or aberrant cell division cycles. In this work, we model this network in a Boolean framework for stochastic simulations. Our model is sufficiently detailed to account for the phenotypes of 40 mutant yeast strains (83% of the experimentally characterized strains that we simulated) and also to simulate an endoreplicating strain (multiple rounds of DNA synthesis without mitosis) and a strain that exhibits 'Cdc14 endocycles' (periodic transitions between metaphase and anaphase). Because our model successfully replicates the observed properties of both wild-type yeast cells and many mutant strains, it provides a reasonable, validated starting point for more comprehensive stochastic-Boolean models of cell cycle controls. Such models may provide a better understanding of cell cycle anomalies in budding yeast and ultimately in mammalian cells., (© 2024. The Author(s).)

Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a metastatic castration-resistant prostate cancer (mCRPC) subtype. It remains unclear, however, whether CDK12 loss drives prostate cancer (PCa) development or uncovers pharmacologic vulnerabilities. Here, we show Cdk12 ablation in murine prostate epithelium is sufficient to induce preneoplastic lesions with lymphocytic infiltration. In allograft-based CRISPR screening, Cdk12 loss associates positively with Trp53 inactivation but negatively with Pten inactivation. Moreover, concurrent Cdk12/Trp53 ablation promotes proliferation of prostate-derived organoids, while Cdk12 knockout in Pten-null mice abrogates prostate tumor growth. In syngeneic systems, Cdk12/Trp53-null allografts exhibit luminal morphology and immune checkpoint blockade sensitivity. Mechanistically, Cdk12 inactivation mediates genomic instability by inducing transcription-replication conflicts. Strikingly, CDK12-mutant organoids and patient-derived xenografts are sensitive to inhibition or degradation of the paralog kinase, CDK13. We therein establish CDK12 as a bona fide tumor suppressor, mechanistically define how CDK12 inactivation causes genomic instability, and advance a therapeutic strategy for CDK12-mutant mCRPC., Competing Interests: Declaration of interests A.M.C. co-founded and serves on scientific advisory boards (SABs) of Lynx Dx, Flamingo Therapeutics, Medsyn Pharma, Oncopia Therapeutics, and Esanik Therapeutics. A.M.C. is an advisor to Aurigene Oncology Limited, Proteovant, Tempus, Rappta, and Ascentage. C.J.L. received research funding from AstraZeneca, Merck KGaA, Artios, and NeoPhore and consultancy, SAB membership, or honoraria payments from FoRx, Syncona, Sun Pharma, Gerson Lehrman Group, Merck KGaA, Vertex, AstraZeneca, Tango, 3rd Rock, Ono Pharma, Artios, Abingworth, Tesselate, Dark Blue Therapeutics, Pontifax, Astex, NeoPhore, Glaxo Smith Kline, and Dawn Bioventures. C.J.L. has stock in Tango, Ovibio, Hysplex, and Tesselate. C.J.L. is named inventor on patents describing use of DNA repair inhibitors and stands to gain from their development and use. J.C. is an advisor for Exai Bio. F.Y.F. has served on SAB or received consulting fees from Astellas, Bayer, Celgene, Clovis Oncology, Janssen, Genentech Roche, Myovant, Roivant, Sanofi, and Blue Earth Diagnostics. F.Y.F. is also an SAB member for Artera, ClearNote Genomics, Serimmune, and BMS (Microenvironment Division). K.D. is an advisor for Kinoteck Therapeutics and has received financial support from Livzon Pharmaceutical Group. Patents for CDK12/13 degraders/inhibitors used here have been filed by the University of Michigan and Shanghai Institute of Organic Chemistry, with A.M.C., K.D., X.W., J.Y., Y. Chang, and J.C.T. as co-inventors., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromosomal DNA replication is a fundamental process of life, involving the assembly of complex machinery and dynamic regulation. In this study, we reconstructed a bacterial replication module (pRC) by artificially clustering 23 genes involved in DNA replication and sequentially deleting these genes from their naturally scattered loci on the chromosome of Escherichia coli. The integration of pRC into the chromosome, moving from positions farther away to close to the replication origin, leads to an enhanced efficiency in DNA synthesis, varying from lower to higher. Strains containing replication modules exhibited increased DNA replication by accelerating the replication fork movement and initiating chromosomal replication earlier in the replication cycle. The minimized module pRC16, containing only replisome and elongation encoding genes, exhibited chromosomal DNA replication efficiency comparable to that of pRC. The replication module demonstrated robust and rapid DNA replication, regardless of growth conditions. Moreover, the replication module is plug-and-play, and integrating it into Mb-sized extrachromosomal plasmids improves their genetic stability. Our findings indicate that DNA replication, being a fundamental life process, can be artificially reconstructed into replication functional modules. This suggests potential applications in DNA replication and the construction of synthetic modular genomes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Finalization of eukaryotic nuclear DNA replication relies on DNA ligase 1 (LIG1) to seal DNA nicks generated during Okazaki Fragment Maturation (OFM). Using a mutational reporter in Saccharomyces cerevisiae, we previously showed that mutation of the high-fidelity magnesium binding site of LIG1 Cdc9 strongly increases the rate of single-base insertions. Here we show that this rate is increased across the nuclear genome, that it is synergistically increased by concomitant loss of DNA mismatch repair (MMR), and that the additions occur in highly specific sequence contexts. These discoveries are all consistent with incorporation of an extra base into the nascent lagging DNA strand that can be corrected by MMR following mutagenic ligation by the Cdc9-EEAA variant. There is a strong preference for insertion of either dGTP or dTTP into 3-5 base pair mononucleotide sequences with stringent flanking nucleotide requirements. The results reveal unique LIG1 Cdc9 -dependent mutational motifs where high fidelity DNA ligation of a subset of OFs is critical for preventing mutagenesis across the genome., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Dysregulated DNA replication is a cause and a consequence of aneuploidy in cancer, yet the interplay between copy number alterations (CNAs), replication timing (RT) and cell cycle dynamics remain understudied in aneuploid tumors. We developed a probabilistic method, PERT, for simultaneous inference of cell-specific replication and copy number states from single-cell whole genome sequencing (scWGS) data. We used PERT to investigate clone-specific RT and proliferation dynamics in  >50,000 cells obtained from aneuploid and clonally heterogeneous cell lines, xenografts and primary cancers. We observed bidirectional relationships between RT and CNAs, with CNAs affecting X-inactivation producing the largest RT shifts. Additionally, we found that clone-specific S-phase enrichment positively correlated with ground-truth proliferation rates in genomically stable but not unstable cells. Together, these results demonstrate robust computational identification of S-phase cells from scWGS data, and highlight the importance of RT and cell cycle properties in studying the genomic evolution of aneuploid tumors., (© 2024. The Author(s).)

Mulberry crinkle leaf virus (MCLV), identified in mulberry plants ( Morus alba L.), is a member of the genus Mulcrilevirus in the family Geminiviridae . The functions of the V2 protein encoded by MCLV remain unclear. Here, Agrobacterium -mediated infectious clones of a wild-type MCLV vII (MCLV WT ) and two V2 mutant MCLV vIIs, including MCLV mV2 (with a mutation of the start codon of the V2 ORF) and MCLV dV2 (5'-end partial deletion of the V2 ORF sequence), were constructed to investigate the roles of V2 both in planta and at the cellular level. Although all three constructs (pCA-1.1MCLV WT , pCA-MCLV mV2 , and pCA-MCLV dV2 ) were able to infect both natural host mulberry plants and experimental tomato plants systematically, the replication of the MCLV mV2 and MCLV dV2 genomes in these hosts was significantly reduced compared to that of MCLV WT . Similarly, the accumulation of MCLV mV2 and MCLV dV2 in protoplasts of Nicotiana benthamiana plants was significantly lower than that of MCLV WT either 24 h or 48 h post-transfection. A complementation experiment further confirmed that the decreased accumulation of MCLV in the protoplasts was due to the absence of V2 expression. These results revealed that MCLV-encoded V2 greatly enhances the level of MCLV DNA accumulation and is designated the replication enhancer protein of MCLV.

Genomic imprinting involves differential DNA methylation and gene expression between homologous paternal and maternal loci. It remains unclear, however, whether DNA replication also shows parent-of-origin-specific patterns at imprinted or other genomic regions. Here, we investigate genome-wide asynchronous DNA replication utilizing uniparental human embryonic stem cells containing either maternal-only (parthenogenetic) or paternal-only (androgenetic) DNA. Four clusters of imprinted genes exhibited differential replication timing based on parent of origin, while the remainder of the genome, 99.82%, showed no significant replication asynchrony between parental origins. Active alleles in imprinted gene clusters replicated earlier than their inactive counterparts. At the Prader-Willi syndrome locus, replication asynchrony spanned virtually the entirety of S phase. Replication asynchrony was carried through differentiation to neuronal precursor cells in a manner consistent with gene expression. This study establishes asynchronous DNA replication as a hallmark of large imprinted gene clusters., Competing Interests: Declaration of interests N.B. is the CSO of NewStem Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Impaired control of the G1/S checkpoint allows initiation of DNA replication under non-permissive conditions. Unscheduled S-phase entry is associated with DNA replication stress, demanding for other checkpoints or cellular pathways to maintain proliferation. Here, we uncovered a requirement for ADARp150 to sustain proliferation of G1/S-checkpoint-defective cells under growth-restricting conditions. Besides its well-established mRNA editing function in inversely oriented short interspersed nuclear elements (SINEs), we found ADARp150 to exert a critical function in mitosis. ADARp150 depletion resulted in tetraploidization, impeding cell proliferation in mitogen-deprived conditions. Mechanistically we show that ADAR1 depletion induced aberrant expression of Cyclin B3, which was causative for mitotic failure and whole-genome duplication. Finally, we find that also in vivo ADAR1-depletion-provoked tetraploidization hampers tumor outgrowth., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Tn3 family transposons are a widespread group of replicative transposons, notorious for contributing to the dissemination of antibiotic resistance, particularly the global prevalence of carbapenem resistance. The transposase (TnpA) of these elements catalyzes DNA breakage and rejoining reactions required for transposition. However, the molecular mechanism for target site selection with these elements remains unclear. Here, we identify a QLxxLR motif in N-terminal of Tn3 TnpAs and demonstrate that this motif allows interaction between TnpA of Tn3 family transposon Tn1721 and the host β-sliding clamp (DnaN), the major processivity factor of the DNA replication machinery. The TnpA-DnaN interaction is essential for Tn1721 transposition. Our work unveils a mechanism whereby Tn3 family transposons can bias transposition into certain replisomes through an interaction with the host replication machinery. This study further expands the diversity of mobile elements that use interaction with the host replication machinery to bias integration., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52's ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability., (© 2024. The Author(s).)

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

R-loops, structures that play a crucial role in various biological processes, are integral to gene expression, the maintenance of genome stability, and the formation of epigenomic signatures. When these R-loops are deregulated, they can contribute to the development of serious health conditions, including cancer and neurodegenerative diseases. The detection of R-loops is a complex process that involves several approaches. These include S9.6 antibody- or RNAse H-based immunoprecipitation, non-denaturing bisulfite footprinting, gel electrophoresis, and electron microscopy. Each of these methods offers unique insights into the nature and behavior of R-loops. In our study, we introduce a novel protocol that has been developed based on a single-molecule DNA combing assay. This innovative approach allows for the direct and simultaneous visualization of RNA:DNA hybrids and replication forks, providing a more comprehensive understanding of these structures. Our findings confirm the transcriptional origin of the hybrids, adding to the body of knowledge about their formation. Furthermore, we demonstrate that these hybrids have an inhibitory effect on the progression of replication forks, highlighting their potential impact on DNA replication and cellular function.

Genetic parasites, including viruses and transposons, exploit components from the host for their own replication. However, little is known about virus-transposon interactions within host cells. Here, we discover a strategy where human cytomegalovirus (HCMV) hijacks L1 retrotransposon encoded protein during its replication cycle. HCMV infection upregulates L1 expression by enhancing both the expression of L1-activating transcription factors, YY1 and RUNX3, and the chromatin accessibility of L1 promoter regions. Increased L1 expression, in turn, promotes HCMV replicative fitness. Affinity proteomics reveals UL44, HCMV DNA polymerase subunit, as the most abundant viral binding protein of the L1 ribonucleoprotein (RNP) complex. UL44 directly interacts with L1 ORF2p, inducing DNA damage responses in replicating HCMV compartments. While increased L1-induced mutagenesis is not observed in HCMV for genetic adaptation, the interplay between UL44 and ORF2p accelerates viral DNA replication by alleviating replication stress. Our findings shed light on how HCMV exploits host retrotransposons for enhanced viral fitness., (© 2024. The Author(s).)

Meier-Gorlin syndrome (MGORS) is an autosomal recessive disorder characterized by short stature, microtia, and patellar hypoplasia, and is caused by pathogenic variants of cellular factors involved in the initiation of DNA replication. We previously reported that biallelic variants in GINS3 leading to amino acid changes at position 24 (p.Asp24) cause MGORS. Here, we describe the phenotype of a new individual homozygous for the Asp24Asn variant. We also report the clinical characteristics of an individual harboring a novel homozygous GINS3 variant (Ile25Phe) and features suggestive of MGORS. Modification of the corresponding residue in yeast Psf3 (Val9Phe) compromised S phase progression compared to a humanized Psf3 Val9Ile variant. Expression of Psf3 Val9Phe in yeast also caused sensitivity to elevated temperature and the replicative stress-inducing drug hydroxyurea, confirming partial loss of function of this variant in vivo and allowing us to upgrade the classification of this variant. Taken together, these data validate the critical importance of the GINS DNA replication complex in the molecular etiology of MGORS., (© 2024 The Authors. Clinical Genetics published by John Wiley & Sons Ltd.)

Control of cellular identity requires coordination of developmental programs with environmental factors such as nutrient availability, suggesting that perturbing metabolism can alter cell state. Here, we find that nucleotide depletion and DNA replication stress drive differentiation in human and murine normal and transformed hematopoietic systems, including patient-derived acute myeloid leukemia (AML) xenografts. These cell state transitions begin during S phase and are independent of ATR/ATM checkpoint signaling, double-stranded DNA break formation, and changes in cell cycle length. In systems where differentiation is blocked by oncogenic transcription factor expression, replication stress activates primed regulatory loci and induces lineage-appropriate maturation genes despite the persistence of progenitor programs. Altering the baseline cell state by manipulating transcription factor expression causes replication stress to induce genes specific for alternative lineages. The ability of replication stress to selectively activate primed maturation programs across different contexts suggests a general mechanism by which changes in metabolism can promote lineage-appropriate cell state transitions., Competing Interests: Declaration of interests D.B.S. is a co-founder of and holds equity in Clear Creek Bio. M.G.V.H. is on the scientific advisory board of Agios Pharmaceuticals, iTeos Therapeutics, Drioa Ventures, Sage Therapeutics, Lime Therapeutics, Pretzel Therapeutics, and Auron Therapeutics, and is on the advisory board of Developmental Cell. P.P.H. has consulted for Auron Therapeutics. J.S.W. serves as an advisor to and/or has equity in KSQ Therapeutics, Maze Therapeutics, and 5AM Ventures. J.M.R. consults for Maze Therapeutics, Waypoint Bio, and Third Rock Ventures. I.S.H. reports financial support from Kojin Therapeutics and consulting fees for Ono Pharma USA., (Copyright © 2024 Elsevier Inc. All rights reserved.)

High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:G→T:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hoggard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: Managing high-grade endometrial cancer in Martinique poses significant challenges. The diversity of copy number alterations in high-grade endometrial tumors, often associated with a TP53 mutation, is a key factor complicating treatment. Due to the high incidence of high-grade tumors with poor prognosis, our study aimed to characterize the molecular signature of these tumors within a cohort of 25 high-grade endometrial cases., Methods: We conducted a comprehensive pangenomic analysis to categorize the copy number alterations involved in these tumors. Whole-Exome Sequencing (WES) and Homologous Recombination (HR) analysis were performed. The alterations obtained from the WES were classified into various signatures using the Copy Number Signatures tool available in COSMIC., Results: We identified several signatures that correlated with tumor stage and disctinct prognoses. These signatures all seem to be linked to replication stress, with CCNE1 amplification identified as the primary driver of oncogenesis in over 70% of tumors analyzed., Conclusion: The identification of CCNE1 amplification, which is currently being explored as a therapeutic target in clinical trials, suggests new treatment strategies for high-grade endometrial cancer. This finding holds particular significance for Martinique, where access to care is challenging., (© 2024. The Author(s).)

Genomic instability is not only a hallmark of senescent cells but also a key factor driving cellular senescence, and replication stress is the main source of genomic instability. Defective prelamin A processing caused by lamin A/C (LMNA) or zinc metallopeptidase STE24 (ZMPSTE24) gene mutations results in premature aging. Although previous studies have shown that dysregulated lamin A interferes with DNA replication and causes replication stress, the relationship between lamin A dysfunction and replication stress remains largely unknown. Here, an increase in baseline replication stress and genomic instability is found in prelamin A-expressing cells. Moreover, prelamin A confers hypersensitivity of cells to exogenous replication stress, resulting in decreased cell survival and exacerbated genomic instability. These effects occur because prelamin A promotes MRE11-mediated resection of stalled replication forks. Fanconi anemia (FA) proteins, which play important roles in replication fork maintenance, are downregulated by prelamin A in a retinoblastoma (RB)/E2F-dependent manner. Additionally, prelamin A inhibits the activation of the FA pathway upon replication stress. More importantly, FA pathway downregulation is an upstream event of p53-p21 axis activation during the induction of prelamin A expression. Overall, these findings highlight the critical role of FA pathway dysfunction in driving replication stress-induced genomic instability and cellular senescence in prelamin A-expressing cells., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The splicing factor RNA-binding motif protein 10 (RBM10) is frequently mutated in lung adenocarcinoma (LUAD) (9-25%). Most RBM10 cancer mutations are loss-of-function, correlating with increased tumorigenesis and limiting the efficacy of current LUAD targeted therapies. Remarkably, therapeutic strategies leveraging RBM10 deficiency remain unexplored. Here, we conduct a CRISPR-Cas9 synthetic lethality (SL) screen and identify ~60 RBM10 SL genes, including WEE1 kinase. WEE1 inhibition sensitizes RBM10-deficient LUAD cells in-vitro and in-vivo. Mechanistically, we identify a splicing-independent role of RBM10 in regulating DNA replication fork progression and replication stress response, which underpins RBM10-WEE1 SL. Additionally, RBM10 interacts with active DNA replication forks, relying on DNA Primase Subunit 1 (PRIM1) that synthesizes Okazaki RNA primers. Functionally, we demonstrate that RBM10 serves as an anchor for recruiting Histone Deacetylase 1 (HDAC1) to facilitate H4K16 deacetylation and R-loop homeostasis to maintain replication fork stability. Collectively, our data reveal a role of RBM10 in fine-tuning DNA replication and provide therapeutic arsenal for targeting RBM10-deficient tumors., (© 2024. The Author(s).)

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Khodaverdian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription-replication conflicts and thus act as modulators of genome dynamics., (© 2024 Luna et al.; Published by Cold Spring Harbor Laboratory Press.)

During each cell cycle, the process of DNA replication timing is tightly regulated to ensure the accurate duplication of the genome. The extent and significance of alterations in this process during malignant transformation have not been extensively explored. Here, we assess the impact of altered replication timing (ART) on cancer evolution by analysing replication-timing sequencing of cancer and normal cell lines and 952 whole-genome sequenced lung and breast tumours. We find that 6%-18% of the cancer genome exhibits ART, with regions with a change from early to late replication displaying an increased mutation rate and distinct mutational signatures. Whereas regions changing from late to early replication contain genes with increased expression and present a preponderance of APOBEC3-mediated mutation clusters and associated driver mutations. We demonstrate that ART occurs relatively early during cancer evolution and that ART may have a stronger correlation with mutation acquisition than alterations in chromatin structure., (© 2024. The Author(s).)

We report here the successful labelling of meiotic prophase I DNA synthesis in the flowering plant, Arabidopsis thaliana. Incorporation of the thymidine analogue, EdU, enables visualisation of the footprints of recombinational repair of programmed meiotic DNA double-strand breaks (DSB), with ~400 discrete, SPO11-dependent, EdU-labelled chromosomal foci clearly visible at pachytene and later stages of meiosis. This number equates well with previous estimations of 200-300 DNA double-strand breaks per meiosis in Arabidopsis, confirming the power of this approach to detect the repair of most or all SPO11-dependent meiotic DSB repair events. The chromosomal distribution of these DNA-synthesis foci accords with that of early recombination markers and MLH1, which marks Class I crossover sites. Approximately 10 inter-homologue cross-overs (CO) have been shown to occur in each Arabidopsis male meiosis and, athough very probably under-estimated, an equivalent number of inter-homologue gene conversions (GC) have been described. Thus, at least 90% of meiotic recombination events, and very probably more, have not previously been accessible for analysis. Visual examination of the patterns of the foci on the synapsed pachytene chromosomes corresponds well with expectations from the different mechanisms of meiotic recombination and notably, no evidence for long Break-Induced Replication DNA synthesis tracts was found. Labelling of meiotic prophase I, SPO11-dependent DNA synthesis holds great promise for further understanding of the molecular mechanisms of meiotic recombination, at the heart of reproduction and evolution of eukaryotes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hernández Sánchez-Rebato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Short-Patch Double Illegitimate Recombination (SPDIR) has been recently identified as a rare mutation mechanism. During SPDIR, ectopic DNA single-strands anneal with genomic DNA at microhomologies and get integrated during DNA replication, presumably acting as primers for Okazaki fragments. The resulting microindel mutations are highly variable in size and sequence. In the soil bacterium Acinetobacter baylyi, SPDIR is tightly controlled by genome maintenance functions including RecA. It is thought that RecA scavenges DNA single-strands and renders them unable to anneal. To further elucidate the role of RecA in this process, we investigate the roles of the upstream functions DprA, RecFOR, and RecBCD, all of which load DNA single-strands with RecA. Here we show that all three functions suppress SPDIR mutations in the wildtype to levels below the detection limit. While SPDIR mutations are slightly elevated in the absence of DprA, they are strongly increased in the absence of both DprA and RecA. This SPDIR-avoiding function of DprA is not related to its role in natural transformation. These results suggest a function for DprA in combination with RecA to avoid potentially harmful microindel mutations, and offer an explanation for the ubiquity of dprA in the genomes of naturally non-transformable bacteria., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Bacillus subtilis was engineered to produce circular subgenomes that are directly transmittable to another B. subtilis. The conjugational plasmid pLS20 integrated into the B. subtilis genome supported not only subgenome replication but also transmission to another B. subtilis species. The subgenome system developed in this study completes a streamlined platform from the synthesis to the transmission of giant DNA by B. subtilis., (© 2024 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein-protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies.

We report that the Escherichia coli chromosome includes novel GC-rich genomic structural elements that trigger formation of post-replication gaps upon replisome passage. The two nearly perfect 222 bp repeats, designated Replication Risk Sequences or RRS, are each 650 kb from the terminus sequence dif and flank the Ter macrodomain. RRS sequence and positioning is highly conserved in enterobacteria. At least one RRS appears to be essential unless a 200 kb region encompassing one of them is amplified. The RRS contain a G-quadruplex on the lagging strand which impedes DNA polymerase extension producing lagging strand ssDNA gaps, $ \le$2000 bp long, upon replisome passage. Deletion of both RRS elements has substantial effects on global genome structure and topology. We hypothesize that RRS elements serve as topological relief valves during chromosome replication and segregation. There have been no screens for genomic sequences that trigger transient gap formation. Functional analogs of RRS could be widespread, possibly including some enigmatic G-quadruplexes in eukaryotes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA replication faces challenges from DNA lesions originated from endogenous or exogenous sources of stress, leading to the accumulation of single-stranded DNA (ssDNA) that triggers the activation of the ATR checkpoint response. To complete genome replication in the presence of damaged DNA, cells employ DNA damage tolerance mechanisms that operate not only at stalled replication forks but also at ssDNA gaps originated by repriming of DNA synthesis downstream of lesions. Here, we demonstrate that human cells accumulate post-replicative ssDNA gaps following replicative stress induction. These gaps, initiated by PrimPol repriming and expanded by the long-range resection factors EXO1 and DNA2, constitute the principal origin of the ssDNA signal responsible for ATR activation upon replication stress, in contrast to stalled forks. Strikingly, the loss of EXO1 or DNA2 results in synthetic lethality when combined with BRCA1 deficiency, but not BRCA2. This phenomenon aligns with the observation that BRCA1 alone contributes to the expansion of ssDNA gaps. Remarkably, BRCA1-deficient cells become addicted to the overexpression of EXO1, DNA2 or BLM. This dependence on long-range resection unveils a new vulnerability of BRCA1-mutant tumors, shedding light on potential therapeutic targets for these cancers., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Precise genomic editing through the combination of CRISPR/Cas systems and recombinant adeno-associated virus (rAAV)-delivered homology directed repair (HDR) donor templates represents a powerful approach. However, the challenge of effectively suppressing leaky transcription from the rAAV vector, a phenomenon associated to cytotoxicity, persists. In this study, we demonstrated substantial promoter activities of various homology arms and inverted terminal repeats (ITR). To address this issue, we identified a novel rAAV variant, Y704T, which not only yields high-vector quantities but also effectively suppresses in cis mRNA transcription driven by a robust promoter. The Y704T variant maintains normal functionality in receptor interaction, intracellular trafficking, nuclear entry, uncoating, and second-strand synthesis, while specifically exhibiting defects in transcription. Importantly, this inhibitory effect is found to be independent of ITR, promoter types, and RNA polymerases. Mechanistic studies unveiled the involvement of Valosin Containing Protein (VCP/p97) in capsid-mediated transcription repression. Remarkably, the Y704T variant delivers HDR donor templates without compromising DNA replication ability and homologous recombination efficiency. In summary, our findings enhance the understanding of capsid-regulated transcription and introduce novel avenues for the application of the rAAV-CRISPR/Cas9 system in human gene therapy., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The rates at which mutations accumulate across human cell types vary. To identify causes of this variation, mutations are often decomposed into a combination of the single-base substitution (SBS) "signatures" observed in germline, soma, and tumors, with the idea that each signature corresponds to one or a small number of underlying mutagenic processes. Two such signatures turn out to be ubiquitous across cell types: SBS signature 1, which consists primarily of transitions at methylated CpG sites thought to be caused by spontaneous deamination, and the more diffuse SBS signature 5, which is of unknown etiology. In cancers, the number of mutations attributed to these 2 signatures accumulates linearly with age of diagnosis, and thus the signatures have been termed "clock-like." To better understand this clock-like behavior, we develop a mathematical model that includes DNA replication errors, unrepaired damage, and damage repaired incorrectly. We show that mutational signatures can exhibit clock-like behavior because cell divisions occur at a constant rate and/or because damage rates remain constant over time, and that these distinct sources can be teased apart by comparing cell lineages that divide at different rates. With this goal in mind, we analyze the rate of accumulation of mutations in multiple cell types, including soma as well as male and female germline. We find no detectable increase in SBS signature 1 mutations in neurons and only a very weak increase in mutations assigned to the female germline, but a significant increase with time in rapidly dividing cells, suggesting that SBS signature 1 is driven by rounds of DNA replication occurring at a relatively fixed rate. In contrast, SBS signature 5 increases with time in all cell types, including postmitotic ones, indicating that it accumulates independently of cell divisions; this observation points to errors in DNA repair as the key underlying mechanism. Thus, the two "clock-like" signatures observed across cell types likely have distinct origins, one set by rates of cell division, the other by damage rates., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Spisak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)